The endomembrane system and functional organization of the eukaryotic cell are critically dependent on membrane fusion and fission. The cellular fusion machinery has been studied for decades, but an understanding of the mechanisms underlying membrane fission has lagged far behind. During the past funding period, we developed a novel model membrane system of supported bilayers with excess reservoir, SUPER templates, to reconstitute membrane fission and vesicle formation and discovered that dynamin-1 (Dyn1) alone can catalyze membrane fission. We also solved high-resolution structures of the Dyn1 GTPase domain dimer in the GDP7AlF4-bound transition state and in the GMPPCP-bound form revealing GTPase- driven conformational changes that accompany membrane fission. Findings derived from our unique combination of biochemistry, cell and molecular biology, biophysics and structure biology have led us to propose a two-stage model for Dyn1-catalyzed fission: in stage 1 Dyn1's mechanochemical activities generate highly localized curvature stress and in stage 2, the PH domains form a catalytic center to guide lipid rearrangements and formation of a hemi-fission intermediate. In Aim 1, we propose detailed structure/function analyses to directly test, elaborate and refine this model and to define the mechanisms underlying Dyn1-catalyzed membrane fission. As dynamin is the prototypical member of a growing family of large fusion and fission GTPases, our findings will reveal new paradigms and guide research towards elucidating the fundamental principles governing organelle dynamics and vesicle formation. In addition to its role in fission, we have provided substantial evidence that dynamin also regulates early stages of clathrin- mediated endocytosis (CME). We recently identified specific biochemical properties of Dyn2, the ubiquitously expressed isoform (i.e. its activity is highly sensitive to membrane curvature) that render it ideally suited to monitor early stages of clathrin coated pit (CCP) assembly and maturation and to catalyze fission only after the formation of deeply invaginated CCPs. The studies proposed in Aim 2 will identify intra- and inter- molecular mechanisms that regulate Dyn2 activity. They will also be the first to functionally link the curvature generating abilities of BAR domain and coat proteins with the fission activity of Dyn2 as requirements for vesicle formation in vitro. CME plays a critical role in regulating how cells interact with each other and their environment. Thus, identifying factors and understanding the mechanisms that regulate dynamin will reveal important insights into the regulation of the critical cellular process of CME. PUBLIC HEALTH RELEVANCE: Clathrin-mediated endocytosis is the primary mechanism by which cells communicate with and adapt to changes in their environment, through nutrient uptake, regulation of signaling receptors and remodeling of plasma membrane composition. The GTPase dynamin is the master regulator of CME and drives membrane fission. We will use biochemical, biophysical, and cell biological approaches to elucidate the mechanism of dynamin-catalyzed fission and to identify factors regulating dynamin activity.